Our laboratory has embarked on a systematic analysis of p15INK4b, which in human acute myeloid leukemia (AML) is inactivated more frequently than any other tumor suppressor gene with cell cycle regulatory properties. p15Ink4b is a cyclin-dependent kinase inhibitor and like other Ink4b family members binds to early G1 cyclin-dependent kinases 4 and 6, thereby preventing their interaction with D cyclins. We have recently provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice. First, retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation within the gene's promoter region. In some cases where leukemias did not have hypermethylation in the primary state, the neoplastic cells developed methylation following transplantation in syngeneic mice. The epigenetic change correlated with decreased expression in primary and transplanted leukemias as shown by TaqMan real-time RT-PCR. Furthermore, treatment of a leukemia cell line that had Ink4b hypermethylation, with the methylation inhibitor 5-aza-2' deoxycytidine, resulted in an increase in p15Ink4b mRNA expression. A second type of study carried out in p15Ink4b-deficient mice provided the most convincing data regarding the tumor suppressor role of Ink4b. Use of the mice in a retrovirus-induced leukemogenesis protocol showed that mice heterozygous for Ink4b deficiency had increased susceptibility to myeloid leukemia. Mice were inoculated with retrovirus MOL4070LTR, a unique myeloid disease-inducing virus, which was recently constructed and produced in our laboratory. The retrovirus provided the cooperating events through insertional mutagenesis that allowed the demonstration of the susceptibility to leukemia provided by loss of one allele. Mice deficient in both alleles were not more susceptible than those deficient in one allele, raising the possibility that there are opposing forces to the development of myeloid leukemia in Ink4b null mice. Our laboratory has shown that Ink4b expression at the mRNA level is up-regulated following treatment with cytokines such as interferon (IFN), TGFbeta, and IL-6 that are found in hematopoietic and immune environments. This is an important way that cells are instructed to growth arrest during differentiation. As a part of an investigation into the mechanisms involved in IFNbeta-induced expression of p15Ink4b mRNA in the murine myeloid cell line M1, we determined that both the interferon consensus sequence binding protein (ICSBP)/IRF-8 and the Ets-like protein PU.1 were able to activate transcription from the Ink4b promoter. Our data suggest that binding of both factors to DNA is required in order to stabilize a PU.1/ICSBP binding complex. This regulation of the Ink4b promoter is apparently myeloid-specific, because both ICSBP and PU.1 are myeloid commitment factors. Interestingly, this study on transcriptional regulation of Ink4b provides, at least in part, an explanation for the tumor suppressor activity of ICSBP demonstrated in ICSBP-deficient mice. These mice develop a chronic myelogenous leukemia (CML)-like disease and this is consistant with the finding that a high percentage of human AMLs and CMLs lack ICSBP transcripts. Inhibitors of DNA methylation are promising options for the treatment of human AML and MDS. This is in part because hypermethylation of INK4B is so prevalent in these diseases and has been shown to be reversible. As treatments for reversing aberrant methylation become more effective, it becomes increasingly important to be able to identify those subgroups of AML that would be the more likely to respond to such treatment. Despite categorization of AML based upon cytogenetic abnormalities, there has been no attempt to correlate epigenetic inactivation of INK4b with known translocations. A recent study from our laboratory shows that AML subtypes based on their cytogenetic abnormalities vary dramatically in their frequency of methylation at the INK4B locus. Most notable is the lack of methylation, sufficient to reduce expression, in all leukemias with inv(16). This is in contrast to t(15;17) and t(8;21) associated leukemias, where levels of methylation of 11% to 43 % were found in 88% and 57 %, respectively, of the AML analyzed. Our ongoing studies are aimed at determining genes that collaborate with inactivation of Ink4b, determining the effects of p15Ink4b on normal hematopoiesis, and investigating further regulation of Ink4b transcription through transcription factors and methylation.